US9400282B2 - Method for quantifying protein - Google Patents

Method for quantifying protein Download PDF

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US9400282B2
US9400282B2 US13/383,925 US201013383925A US9400282B2 US 9400282 B2 US9400282 B2 US 9400282B2 US 201013383925 A US201013383925 A US 201013383925A US 9400282 B2 US9400282 B2 US 9400282B2
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peptides
proteins
standard
mrm
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Keiichi Nakayama
Masaki Matsumoto
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Kyushu University NUC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

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  • the present invention relates to a method for quantifying protein by a multiple reaction monitoring (MRM) method. More specifically, the present invention relates to a method useful for measuring the absolute amount of a target protein contained in a protein mixture, such as a biological sample.
  • MRM multiple reaction monitoring
  • the homeostasis of life is maintained by a network composed of numerous proteins. Since various diseases can be perceived as a failure of homeostasis, the amount of each protein present in a living body can be used as a biomarker for elucidation of the molecular mechanism or diagnosis of diseases, or as a criterion for therapeutic prognosis. Although both high comprehensiveness and high sensitivity are required in protein detection and quantification to achieve this purpose, there is presently no technique that is sufficient for practical use.
  • Examples of known high-sensitivity protein detection methods include immunochemical methods, such as western blotting utilizing antibodies specific to individual proteins.
  • immunochemical methods such as western blotting utilizing antibodies specific to individual proteins.
  • the acquisition of specific antibodies is a prerequisite, and the obtained results greatly depend on the quality of the antibody used.
  • all antibodies against individual proteins must be prepared, and analysis must be repeated using each individual antibody. However, making such comprehensive analysis is substantially impossible.
  • Examples of known methods for comprehensive analysis of proteins include proteome analysis methods using a mass spectrometer as basic technology.
  • proteome analysis methods using a mass spectrometer as basic technology.
  • detecting trace proteins is extremely difficult.
  • two-dimensional electrophoresis which is a typical proteomic expression analysis method, can only detect proteins with high expression levels.
  • quantitative shotgun proteomics which is a combination of LC-MS/MS with stable isotope labeling (SILAC, ICAT, iTRAQ)
  • the number of proteins that can be detected is only several hundreds to about 3,000.
  • detecting or quantifying trace proteins is impossible (see Non-patent Literature (NPL) 1 to 3).
  • NPL Non-patent Literature
  • the purpose of these methods is generally relative quantification, rather than absolute quantification, of proteins. It is thus difficult to derive a quantitative relationship between proteins by comparing quantitative values of the proteins individually obtained in different research and testing laboratories by using such methods.
  • MRM multiple reaction monitoring
  • SRM selective reaction monitoring
  • the number of peptides generated from one protein by enzymatic digestion, etc. may range from several tens to several hundreds.
  • Selection of the target peptide to be subjected to MRM is a very important step in specifying the sensitivity of this method.
  • the selection of the target peptide for MRM is made by utilizing a measured spectrum obtained by shotgun proteome analysis; or depends on a method of theoretical estimation under specific conditions.
  • the MRM method is currently recognized only as a methodology for reviewing and confirming the results obtained by shotgun proteome analysis, etc., and is not expected to be used as a large-scale screening method.
  • an object of the present invention is to provide a method for measuring the absolute amounts of target proteins contained in a biological sample that is a mixture of various proteins, and more preferably a method that can measure the absolute amounts of target proteins at a high speed.
  • the MRM method which has been used as a means for analyzing a specific protein, can be converted into an ultra-high-sensitivity large-scale protein analysis method by utilizing genome information resources that have recently become abundant.
  • MRM is a method for specifically quantifying a specific component contained in a complicated sample by providing a combination of two mass filters (MRM transitions: a combination of mass (m/z) filters Q1 and Q3), i.e., a mass filter (Q1) through which an ion having a specific mass (a peptide herein) can be passed, and a mass filter (Q3) through which fragments generated by gas collision-induced dissociation (CID) can be passed, and detecting an ion (a peptide) that can pass through these two mass filters (Non-patent Literature (NPL) 5).
  • MRM transitions a combination of mass (m/z) filters Q1 and Q3
  • a mass filter (Q1) through which an ion having a specific mass (a peptide herein) can be passed
  • CID gas collision-induced dissociation
  • MRM analysis is expected as a new technique that can solve the broad dynamic range problem of the proteome.
  • mass data MS/MS spectrum
  • MS/MS spectrum data obtained by shotgun proteomics analysis are generally used as such information, MRM analysis of trace proteins that cannot be detected by shotgun analysis is difficult. As a means for solving these problems, in particular the latter problem, the present invention was accomplished.
  • Item 1 A method for identifying and quantifying target proteins in a test sample containing plural kinds of proteins, the method comprising:
  • step (B) a step of adding, to the sample obtained in step (A), known amounts of internal standards that are prepared by fragmenting standard proteins that are identical to the target proteins and labeling the fragmented-standard proteins with a stable isotope Y;
  • step (C) a step of placing the sample obtained in step (B) in an LC-MS/MS device, and performing multiple reaction monitoring (MRM) analysis using MRM transitions selected for the internal standards;
  • step (D) a step of identifying, in the MRM chromatogram detected in step (C), peptides derived from the target proteins (target peptides) that show the same retention time as peptides derived from the internal standards (peptides of the internal standards), and quantifying the target proteins in the test sample by comparing the peak areas of the peptides of the internal standards with the peak areas of the target peptides.
  • Item 2 The method according to Item 1, wherein the MRM transitions are selected by a method comprising:
  • step (2) a step of placing the internal standards obtained in step (1) in the LC-MS/MS device and determining the peptide ion intensities of fragments corresponding to the peptides derived from the standard proteins;
  • step (C) or (D) is performed based on a calibration reference table of the standard proteins.
  • step (C) or (D) is performed based on a calibration reference table of the standard proteins.
  • step (C) or (D) is performed based on a calibration reference table of the standard proteins.
  • step (C) or (D) is performed based on a calibration reference table of the standard proteins.
  • the calibration reference table of each standard protein is prepared by a method comprising:
  • step (b) a step of placing the internal standard obtained in step (a) in the LC-MS/MS device and performing multiple reaction monitoring (MRM) analysis using the MRM transitions selected for the internal standard;
  • MRM multiple reaction monitoring
  • step (d) storing the list prepared in step (c) for use as the calibration reference table of the standard protein.
  • Item 5 The method according to Item 4, wherein steps (a) to (d) are repeated using two or more standard proteins so as to prepare and store calibration reference tables of the standard proteins, thus constructing a standard protein library.
  • peptide fragments obtained by digestion treatment of proteins are quantified, and proteins are not directly quantified. Accordingly, the quantification is not affected by the solubility of protein molecules, and proteins having various chemical properties can be treated in the same manner.
  • a mass spectrometer is used for the measurement. Accordingly, proteins in the amount of several fmol can be quantified.
  • FIG. 1 shows the manner of selecting MRM transitions using PFTS in Example 1.
  • FIG. 1A shows the results of LC-MS/MS analysis of PFTS (mTRAQ-117-labeled p27 Kip1 digest) in data-dependent automatic MS/MS acquisition mode. The horizontal axis represents time (min), and the vertical axis represents the strength (cps). However, specific numerals themselves are not particularly relevant to the invention.
  • FIG. 1B shows the results obtained by database-searching the data obtained in FIG. 1A , using MASCOT and ProteinPilot, to select MRM transitions; and actually subjecting PFTS (mTRAQ-117-labeled p27 Kip1 digest) as a sample to MRM analysis using the selected MRM transitions.
  • FIG. 2 shows a calibration curve prepared using PFTS.
  • the calibration curve was obtained by calculating the area ratio of the mTRAQ-113 p27 Kip1 digest to the mTRAQ-117 p27 Kip1 digest, and plotting the calculation results relative to the concentration of the mTRAQ-113-p27 Kip1 digest.
  • FIG. 3 shows the results obtained by preparing, as model cells, Skp2 knockdown HeLa cells (Skp2KD1-2 cells and Skp2KD3-10 cells), and measuring the amount of p27 Kip1 in the cells by western blotting using a recombinant p27 Kip1 as a control.
  • FIG. 4 shows the results of MRM analysis performed using p27 Kip1 MRM transitions (two types of peptides) prepared using PFTS (mTRAQ-117-labeled p27 Kip1 digest) and calibration curves in Example 1, and comparison of the amount (absolute amount) of p27 Kip1 present in Skp2 knockdown cells (Skp2KD1-2 cells and Skp2KD3-10 cells) with that in the control cells (Mock).
  • FIG. 4A shows a chromatogram of the MRM analysis. The horizontal axis represents time (minute), and the vertical axis represents intensity (cps). However, specific numerals themselves are not particularly relevant to the invention.
  • FIG. 4 shows the results of MRM analysis performed using p27 Kip1 MRM transitions (two types of peptides) prepared using PFTS (mTRAQ-117-labeled p27 Kip1 digest) and calibration curves in Example 1, and comparison of the amount (absolute amount) of p27 Kip1 present
  • FIG. 4B shows the amount of p27 Kip1 contained in each of the cell extracts (control cells (Mock), knockdown cells (Skp2 KD 1-2 cells), and knockdown cells (Skp2 KD 3-10 cells)) (20 ⁇ g each).
  • arrow a indicates a peak derived from the sample
  • arrow b indicates a peak derived from the internal standard.
  • the method of the present invention identifies and quantifies target proteins in a sample containing plural kinds of proteins.
  • a feature of this method is that the method comprises the following steps (A) to (D):
  • step (B) a step of adding, to the sample obtained in step (A), known amounts of internal standards that are prepared by fragmenting a standard proteins that are identical to the target proteins, and labeling the fragmented standard proteins with a stable isotope Y;
  • step (C) a step of placing the sample obtained in step (B) in an LC-MS/MS device, and performing multiple reaction monitoring (MRM) analysis using MRM transitions selected for the internal standards;
  • step (D) a step of identifying, in the MRM chromatogram detected in step (C), peptides derived from the target proteins (target peptides) that show the same retention time as peptides derived from the internal standards (peptides of the internal standards), and quantifying the target protein in the test sample by comparing the peak areas of the peptides of the internal standards with the peak areas of the target peptides.
  • test sample to be subjected to the method of the present invention is not particularly limited, and may be anything that contains two or more proteins.
  • samples include samples (biological samples) derived from microorganisms, plants, and animals (including humans). Specific examples thereof include blood, urine, saliva, hair, cells, cell tissues, and treated products thereof; and protein-containing samples prepared by gene recombination techniques.
  • step (A) the target proteins are fragmented to each collection of peptides, and the fragmented target proteins are subsequently labeled with a stable isotope X.
  • proteolytic enzyme such as trypsin
  • chemical cleavage methods such as a method using cyanogen bromide
  • Digestion by protease is preferable. It is known that a given mole quantity of protein produces the same mole quantity for each tryptic peptide cleavage product if the proteolytic digest is allowed to proceed to completion. Thus, determining the mole quantity of tryptic peptide to a given protein allows determination of the mole quantity of the original protein in the sample.
  • Absolute quantification of the target proteins can be accomplished by determining the absolute amount of the target protein-derived peptides contained in the protease digestion (collection of peptides). Accordingly, in order to allow the proteolytic digest to proceed to completion, reduction and alkylation treatments are preferably performed before protease digestion with trypsin to reduce and alkylate the disulfide bonds contained in the target proteins.
  • the obtained digest (collection of peptides) is subjected to labeling with a stable isotope X.
  • stable isotopes X include 1 H and 2 H for hydrogen atoms, 12 C and 13 C for carbon atoms, and 14 N and 15 N for nitrogen atoms. Any isotope can be suitably selected therefrom.
  • Labeling by a stable isotope X can be performed by reacting the digest (collection of peptides) with a reagent containing the stable isotope.
  • reagents Preferable examples of such reagents that are commercially available include mTRAQ (registered trademark) (produced by Applied Biosystems), which is an amine-specific stable isotope reagent kit.
  • mTRAQ is composed of 2 or 3 types of reagents (mTRAQ-light and mTRAQ-heavy; or mTRAQ-D0, mTRAQ-D4, and mTRAQ-D8) that have a constant mass difference between them as a result of isotope-labeling, and that are bound to the N-terminus of a peptide or the primary amine of a lysine residue.
  • step (B) a known amount of internal standards are added to the sample obtained in step (A).
  • the internal standards used herein are digests (collections of peptides) obtained by fragmenting proteins (standard proteins) consisting of the same amino acid sequence as the target proteins to be measured, and labeling the obtained digests (collections of peptides) with a stable isotope Y.
  • the fragmentation treatment can be performed in the same manner as above for the target proteins. Labeling with a stable isotope Y can also be performed in the same manner as above for the target protein.
  • the stable isotope Y used herein must be an isotope that has a mass different from that of the stable isotope X used for labeling the target protein digest.
  • mTRAQ registered trademark
  • mTRAQ-heavy should be used to label digests of standard proteins.
  • step (C) the sample obtained in step (B) is first placed in an LC-MS/MS device, and then multiple reaction monitoring (MRM) analysis is performed using MRM transitions selected for the internal standards.
  • MRM reaction monitoring
  • the sample (collections of peptides labeled with a stable isotope) obtained in step (B) is separated first by one-dimensional or multi-dimensional high-performance liquid chromatography.
  • liquid chromatography examples include cation exchange chromatography, in which separation is conducted by utilizing electric charge difference between peptides; and reversed-phase chromatography, in which separation is conducted by utilizing hydrophobicity difference between peptides. Both of these methods may be used in combination.
  • each of the separated peptides is subjected to tandem mass spectrometry (MS/MS) by using a tandem mass spectrometer (MS/MS spectrometer) comprising two mass spectrometers connected in series.
  • MS/MS spectrometer enables the detection of several fmol levels of a target protein.
  • MS/MS analysis enables the analysis of internal sequence information on peptides, thus enabling identification without false positives.
  • an electrospray ionization method which is a soft ionizing method, is preferably used as the ionization method.
  • analyzers include magnetic sector mass spectrometers (Sector MS), quadrupole mass spectrometers (QMS), time-of-flight mass spectrometers (TOFMS), and Fourier transform ion cyclotron resonance mass spectrometers (FT-ICRMS); a combination of these spectrometers may also be used as the analyzer.
  • Sample MS magnetic sector mass spectrometers
  • QMS quadrupole mass spectrometers
  • TOFMS time-of-flight mass spectrometers
  • FT-ICRMS Fourier transform ion cyclotron resonance mass spectrometers
  • a feature of the method of the present invention is that to select the target peptides, multiple reaction monitoring (MRM) analysis is performed by using MRM transitions selected for the internal standards.
  • MRM multiple reaction monitoring
  • the selection of MRM transitions and selection of a target peptides using the MRM transitions can be performed by a method comprising the following steps (1) to (3):
  • step (2) a step of placing the internal standards obtained in step (1) in an LC-MS/MS device, and determining the peptide ion intensities corresponding to the peptides derived from the standard proteins;
  • Steps (1) and (2) are carried out by digesting recombinant proteins (standard proteins) consisting of an amino acid sequence identical to that of the target proteins, with a protease such as trypsin, as described above, and subsequently labeling the digest with a stable isotope (such as mTRAQ heavy: labeled with IS); and conducting measurement by LC-MS in an automatic MS/MS solution mode using the labeled digest as a precursor-fragment transition selection standard (PFTS).
  • the standard proteins are preferably recombinant proteins prepared based on a full-length cDNA library.
  • the obtained data are put through a search engine to perform spectral assignment and to list the peptides experimentally detected for each protein.
  • the detected peptides are grouped for each protein, and three or more fragments having an m/z value larger than that of the precursor ion and three or more fragments with an m/z value of 500 or more are selected from each MS/MS spectrum in descending order of signal intensity on the spectrum. From these, two or more fragments are selected in descending order of intensities, and the average of the strength is defined as the expected sensitivity of the MRM transitions.
  • PFTS may consist of a single purified recombinant protein, or a mixture of plural proteins.
  • the peptides with high expected sensitivity are preferably obtained with good reproducibility in order to prevent calculation error between columns in elution time (retention time) on the chromatogram (e.g., reversed-phase chromatogram) in liquid chromatography. More specifically, it is preferable that the chromatogram be normalized by using the elution time of two or more standard peptides, and that the elution time calculation error be controlled to within ⁇ 1 minute. This allows elution time-dependent MRM analysis (scheduled MRM analysis), and the number of proteins that can be quantified per analysis is drastically increased (approximately 400 proteins per run).
  • Step (D) Quantification of the Target Protein in the Test Sample
  • Step (D) comprises identifying, in the MRM chromatogram detected in step (C), peptides derived from the target proteins (target peptides) that show the same retention time as peptides derived from the internal standards (peptides of the internal standards), and quantifying the target protein in the test sample by comparing the peak areas of the internal standard peptides with the peak areas of the target peptides.
  • the target proteins can be quantified by utilizing a calibration curve of the standard proteins prepared beforehand.
  • the calibration curve can be prepared by the following method.
  • a recombinant protein consisting of an amino acid sequence that is identical to that of the target protein (a standard protein) is digested with a protease such as trypsin, as described above.
  • a protease such as trypsin
  • precursor-fragment transition selection standards (PFTS) of a known concentration are individually labeled with two different types of stable isotopes (i.e., one is labeled with a stable isomer used to label an internal standard peptide (labeled with IS), whereas the other is labeled with a stable isomer used to label a target peptide (labeled with T)).
  • PFTS precursor-fragment transition selection standards
  • Plural samples are produced by blending a certain amount of the IS-labeled PTFS with various concentrations of the T-labeled PTFS. These samples are placed in the aforementioned LC-MS/MS device to perform MRM analysis. The area ratio of the T-labeled PTFS to the IS-labeled PTFS (T-labeled PTFS/IS-labeled PTFS) on the obtained MRM chromatogram is plotted against the amount of the T-labeled PTFS to prepare a calibration curve (see FIG. 2 ).
  • the absolute amount of the target protein contained in the test sample can be calculated.
  • calibration reference tables regarding MRM transitions, standard peptides derived from the standard proteins, and retention time, etc., of and the standard peptides in LC-MS/MS are preferably prepared beforehand.
  • Such calibration reference tables are also preferably prepared beforehand for the standard proteins corresponding to individual target proteins. In this case, with reference to such calibration reference tables and calibration curves, steps (C) and (D) can be performed.
  • the calibration reference tables can be prepared by the method comprising the following steps:
  • step (b) a step of placing the internal standard obtained in step (a) in an LC-MS/MS device, and performing multiple reaction monitoring (MRM) analysis using MRM transitions selected for the internal standard;
  • MRM multiple reaction monitoring
  • step (d) a step of storing the list prepared in step (c) for use as a calibration reference table of the standard protein.
  • a library of standard proteins may be constructed by repeating steps (a) to (d) using at least two standard proteins, and preparing and storing calibration reference tables of the individual standard proteins. With the construction of such a library, the method of the present invention can determine absolute amounts of all of the proteins.
  • the present invention is directed to a method for measuring the amounts of proteins.
  • synthesis and use of internal standard peptides corresponding to sites subjected to post-translational modification, such as phosphorylation, glycosylation, nitration, or citrullination, as peptides containing a post-translational modification site would enable measurement of the amounts of post-translationally modified target proteins.
  • synthesis and use of internal standard peptides corresponding to peptides present in a living body would enable measurement of the amount of the peptide.
  • p27 Kip1 which is a human cyclin-dependent kinase inhibitor protein
  • pGEX-6P1 E. coli expression vector was incorporated into a pGEX-6P1 E. coli expression vector, and expressed as a GST-fusion protein in E. coli .
  • the GST-fused p27 Kip1 was bound to Glutathione Sepharose, and the GST moiety was cleaved and eluted by treatment with PreScission Protease (produced by GE Healthcare).
  • PreScission Protease produced by GE Healthcare
  • the purified p27 Kip1 (600 ng) prepared above was dissolved in 100 ⁇ l of 100 mM Tris-HCl (pH 8.5) containing 7M guanidine hydrochloride, and incubated at 86° C. for 1 hour. After quenching, the resulting solution was mixed with an equal amount of 100 mM Tris-HCl (pH 8.5), and 0.1 ⁇ g of lysyl endopeptidase was added to perform incubation (37° C.) overnight.
  • the obtained product (p27 Kip1 digest) was desalted using Sep-PAK C18 (produced by Japan Waters Co., Ltd.) (50 mg), and then subjected to centrifugal concentration.
  • the dried product (p27 Kip1 digest) obtained by centrifugal concentration was re-dissolved in 40 ⁇ l of iTRAQ buffer (supplied with an iTRAQ reagent kit, produced by Applied Biosystems), and divided equally between two tubes.
  • the solution in the tubes was treated with mTRAQ-light (mTRAQ-113: 1 unit) or mTRAQ-heavy (mTRAQ-117: 1 unit) at room temperature for 2 hours for labeling. After 100 ⁇ l of ultrapure water was added to each and mixed, the resulting mixtures were allowed to stand at room temperature for 1 hour, and then subjected to centrifugal concentration again.
  • the concentrates were each re-dissolved in 20 ⁇ l of a 0.5% aqueous trifluoroacetic acid solution.
  • the solutions thus obtained were used as stock samples (mTRAQ-113-labeled p27 Kip1 digest and mTRAQ-117-labeled p27 Kip1 digest: 10 pmol/ ⁇ l each).
  • the p27 Kip1 digest labeled with mTRAQ-heavy (mTRAQ-117) was used as a precursor-fragment transition selection standard (PFTS).
  • Twenty fmol of the digest was subjected to LC-MS/MS analysis (IDA mode) using a triple quadrupole mass spectrometry system (QTRAP 5500: produced by AB/Sciex) comprising a multi-dimensional chromatography HPLC system (Paradigm MS2, produced by Michrom BioResources, Inc.) and an autosampler (HTS-PAL, produced by HCT) connected to each other.
  • the measurement was made using an L-column (produced by Chemicals Evaluation and Research Institute, filler diameter: 3 ⁇ m, inner diameter: 100 ⁇ m, column length: 15 cm) under the following conditions:
  • Mobile phase A 0.1% formic acid/2 volume % aqueous methanol solution
  • Mobile phase B 0.1% formic acid/98% aqueous methanol solution
  • FIG. 1A shows the results.
  • the obtained data were searched against human IPI version 3.1.6, which is a human protein database, using the database search engine MASCOT with ProteinPilot software (produced by Applied Biosystems).
  • MRM transitions Ten types of peptides with high expected sensitivity were selected from the peptides identified by the search. For each peptide, at least two types of fragment ions (MRM transitions) were selected. Using the selected MRM transitions, an mTRAQ-117-labeled p27 Kip1 digest, which was used as a sample, was actually subjected to MRM analysis again.
  • FIG. 1B shows the results.
  • the obtained MRM chromatogram revealed that two types of peptides (indicated by arrows in FIG. 1B ) can be detected with high sensitivity.
  • high sensitivity MRM transition configuration information and retention time were recorded.
  • the mTRAQ-117-labeled p27 Kip1 digest and the mTRAQ-113-labeled p27 Kip1 digest prepared in (3) were mixed at the various ratios shown in Table 1, and 1 ⁇ l of each mixture was subjected to MRM analysis.
  • the obtained MRM chromatograms were analyzed using MultiQuant (Applied Biosystems) to calculate the area ratio of the mTRAQ-113-p27 Kip1 digest to the mTRAQ-117-p27 Kip1 digest.
  • the area ratio was plotted against the concentration of the mTRAQ-113-p27 Kip1 digest to prepare a calibration curve, which was stored as a MultiQuant calibration curve file (see FIG. 2 ).
  • FIG. 2 shows that the calibration curve is a straight line, thus confirming guaranteed reliability of the quantification.
  • the Skp2 protein is a ubiquitination enzyme of p27 Kip1 (a human cyclin-dependent kinase inhibitor protein) prepared in Example 1.
  • FIG. 3 shows the results.
  • the amount of p27 Kip1 present in 20 ⁇ g of each extract was estimated. It was confirmed that the amount of p27 Kip1 in the control cells (Mock) was 3 fmol; the amount of p27 Kip1 in the knockdown cells (Skp2 KD 1-2 cells) was 20 fmol; and the amount of p27 Kip1 in the knockdown cells (Skp2 KD 3-10 cells) was 10 fmol.
  • the obtained products were desalted using Sep-PAK C18 (produced by Japan Waters, Co., Ltd.) (50 mg), and then subjected to centrifugal concentration.
  • the dried products (digests) obtained by centrifugal concentration were re-dissolved in 20 ⁇ l of iTRAQ buffer (supplied with an iTRAQ reagent kit, produced by Applied Biosystems), and treated with mTRAQ-light (mTRAQ-113) (1 unit) at room temperature for 2 hours. After 100 ⁇ l of ultrapure water was added to each and mixed, the resulting mixtures were allowed to stand at room temperature for 1 hour and then subjected to centrifugal concentration again.
  • iTRAQ buffer supplied with an iTRAQ reagent kit, produced by Applied Biosystems
  • mTRAQ-113 mTRAQ-light
  • the concentrates were each re-dissolved in 20 ⁇ l of a 0.5% aqueous trifluoroacetic acid solution, and 500 fmol of p27 Kip1 -heavy (a mTRAQ-117-labeled p27 Kip1 digest) was added as an internal standard.
  • the mixtures were desalted using a reversed-phase microcolumn, and subjected to centrifugal concentration.
  • FIGS. 4A and 4B show the results.
  • sample-derived peaks that completely overlapped with internal standard (p27 Kip1 -heavy)-derived MRM peaks were detected.
  • the actual measurement data were read by MultiQuant (produced by Applied Biosystems) to calculate the area ratio, and the calibration curve obtained in step 4 was recalled to calculate the absolute amount of p27 Kip1 -light (p27 Kip1 -113) in the sample. As shown in FIG.
  • the results show that the amount of p27 Kip1 in 20 ⁇ g of each of the cell extracts was such that the amount of p27 Kip1 in the control cells (Mock) was 3 fmol; the amount of p27 Kip1 in the knockdown cells (Skp2 KD 1-2 cells) was about 17 fmol; and the amount of p27 Kip1 in the knockdown cells (Skp2 KD 3-10 cells) was about 11 fmol, which were thus confirmed to be substantially the same as the estimates.

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